Diarrhea from foodborne pathogens is estimated to cause 47 million illnesses/year in the USA alone. Given the lack of specificity of symptoms and limitations of microbiological assays, the etiologic agent is often not identified. Recently, a multiplex PCR assay (FilmArray GI Panel (GIP), BioFire Diagnostics, Salt Lake City, UT) was approved that screens diarrheal stool samples for 22 enteric pathogens, providing a new perspective on enteric pathogens in the USA. Particularly surprising is the prevalence of enteropathogenic E. coli (EPEC), ranging from 7-22% of diarrhea samples. EPEC dogma states it is a pathogen of infants in developing countries who are often malnourished. EPEC is non-invasive; instead, EPEC pathogenesis depends on a type three secretion system that injects effector proteins into host cells inducing actin pedestals, attaching/effacing lesions, and physiological changes in intestinal epithelial cells that contribute to diarrhea including alterations in ion transporters and tight junctions. The presence or absence of the EPEC adherence factor plasmid defines typical EPEC (tEPEC) and atypical EPEC (aEPEC), respectively. GIP does not distinguish between tEPEC and aEPEC nor does it detect other virulence genes such as known adhesins or toxins, raising the question of the virulence potential of GIP- eae-positive strains. Despite the abundance of epidemiological data of EPEC infections worldwide, there is a lack of knowledge about the characteristics of EPEC infections in industrialized nations. Also, while there is a wealth of information regarding EPEC T3SS and effectors in prototypical EPEC strains such as E2348/69, there is a dearth of information linking clinical EPEC strains with functional virulence assays or diarrheal severity. Although EPEC is detected in some healthy individuals and is often part of a mixed infection, aEPEC occurrence is on the rise in developing and developed nations and determined to be the causative agent for many outbreaks in both children and adults. EPEC is also often detected in patients who are immunocompromised due to cancer. Given the frequency of EPEC identification, the uncertainty of its contribution to diarrhea, and the lack of detection of other EPEC virulence genes by GIP, it is prudent to characterize these strains to gain insight into their pathogenicity. Therefore, the overarching goal of this proposal is to determine the virulence properties of EPEC strains identified by GIP and isolated from humans with diarrhea. The specific aims of this proposal are: 1) Compare EPEC bacterial loads in samples from single versus mixed infections as identified by GIP and correlate bacterial burden with diarrheal severity; 2) Isolate and characterize genomic virulence propensity of eae-positive colonies from EPEC-GIP-positive stool samples; 3) Characterize the phenotypic virulence properties of isolated EPEC strains related to attachment, disruption of tight junctions (TJ), and Cl-/HCO3- exchanger (DRA) inhibition.